HLA (Human Leukocyte Antigen) that is Human major histocompatibility antigen, is expressed on membranes of imuunocompetent cells, presents processed peptides derived from exogenous and endogenous antigens to T lymphocytes, and functions as a marker to recognize self and non-self. The present invention relates to a method, a reagent and a kit for typing of the HLA class I alleles. This invention is especially useful for judgement of compatibility between a donor and a recipient in organ transplantation, and for association analysis between the HLA class I genes and various types of diseases in the clinical and medical field. This invention enables us to easily automate and mechanize detection and determination of the HLA class I alleles.
Typing of the HLA antigens has been mainly performed by the serological method using human alloantibodies. By using the specific antibodies to each HLA antigen which are contained in cord blood or serum from subjects who have frequently undergone blood transfusion, complement-mediated cytotoxicity is caused in the antigen-antibody reaction. It changes permeability of positive cell membranes to take an eosinic pigment into the cell, resulting in being detected as colored and expanding cells with a microscope. It is possible to type HLA-A, HLA-B and HLA-C antigens belonging to HLA class I, and HLA-DR and HLA-DQ antigens belonging to HLA class II by this method. However, this method has problems in terms of collection, quality control and supply of the specific antibodies. Furthermore, the survival rate of cells is utilized as an indicator for judgement in this method. Therefore, poor conditions of subjects, for example, a low survival rate of cells caused by disease or influence by passage of time after blood collection, lead to decrease of credibility for results of testing.
In recent years, a development of molecular biotechnology has enabled us to analyze the region of genes encoding the HLA antigens. That has clarified the correspondence between the HLA antigens and the sequences of the HLA genes. This means it has been possible to identify the HLA antigen type by analyzing the specific sequences of the HLA genes (DNA typing). Especially, PCR (polymerase chain reaction) method which can high-sensitively detect a slight change of sequences isutilized to type the HLA-DR, -DQ, or -DP genes belonging to HLA class II. Several PCR-based typing methods for HLA class II DNA such as PCR-SSOP (Sequence-Specific Oligonucleotide Probe) method, PCR-RFLP (Restriction Fragment Length Polymorphism) method, PCR-SSP (Sequence-Specific Primers) method and PCR-SSCP (Single Strand Conformation Polymorphism) method have been developed. In all these methods, the gene region to analyze is amplified by the PCR method and then the variable region in the sequences of the amplified products is analyzed by combination with another methods in order to distinguish the genotype. The HLA class II DNA typing method makes it possible to classify the HLA type at the allele level in addition to classification by the classical serological method using human alloantisera.
Development of the PCR based-method for BLA class I DNA typing is delayed remarkably, comparing with HLA class II typing. The reasons are as follows: (1) While almost all the class II gene mutations (gene substitutions), including those which reflect the specificity of antigens, concentrate in the region of the exon 2, the class I gene mutations are interspersed among the regions of the exons 2 and 3, or the exon 4. (2) The HLA class I genes, including non-classical genes (HLA-E, -F and -G) and pseudogenes (HLA-H, -J, -K and -L), are highly homologous among them.
To date, several HLA class I DNA typing methods have been reported. However, all these methods require complicated manipulation, strict reaction condition and skill. Those are not suitable for handling a large number of samples and offer only low resolution HLA typing. Furthermore, the typing methods for each gene are not standardized.
The purpose of this invention is to solve problems of the manipulation of HLA class I locus antigen typing by the classical serological method, and to prodive a method, a kit and a reagent for classifing the subtype of the HLA class I antigens at the allele level (allele typing), which has not been distinguished by the classical method. Furthermore, the aim of this invention is to provide a method for typing of the HLA class I alleles which can automate and machanize easily.
As a result of intensive studies for these subjects, the inventors have established primers which can amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles and specific primers to the common sequences among all alleles in the group consisting of the specific HLA-A alleles or the specific HLA-B alleles. The inventors have established probes which can specifically hybridize with the sequence of at least one specific HLA-A allele, at least one specific BLA-B allele or at least one specific HLA-C allele. The inventors have found out that it is possible to distinguish the HLA class I antigen or allele, by hybridizing the PCR amplified products derived from the specific HLA class I allele or the specific group with the DNA probes described above which are immobilized on wells of microtiter plates, adding an enzyme-conjugate which can specifically bond to a label of the amplified products at the same time as or after the hybridization, and adding a chromogenic substrate, a luminescent substrate or a fluorescent substrate to the mixture, to detect as signals whether or not the amplified products are hybridized with the immobilized DNA probes. Thus, they have accomplished this invention.
The main embodiment of this invention is a method for typing of HLA class I alleles, which comprises the following steps from (a) to (d).
(a) A step, using HLA class I gene or nucleic acids containing their fragment for a template,
(1) To non-selectively amplify all HLA-A alleles, all HLA-B alleles or all HLA-C alleles by a PCR method using a primer pair which can amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles, or
(2) To selectively amplify a specific group consisting of specific HLA-A alleles or specific HLA-B alleles by a PCR method using a primer pair which is specific to the common sequence to alleles of the specific group consisting of the specific HLA-A alleles or the specific HLA-B alleles,
(b) A step to add the above products amplified by the PCR method to wells of microtiter plates, wherein each well is modified with a carboxyl group to covalently immobilize amino-modified DNA probes which can specifically hybridize with the sequence of at least one specific HLA-A allele, at least one specific HLA-B allele or at least one specific HLA-C allele, and to hybridize the amplified products with the immobilized DNA probes, wherein the DNA probes are selected depending on the above amplified specific HLA class I gene or group;
(c) A step to detect as signals whether or not the amplified products are hybridized with the immobilized probes; and
(d) A step to determine the type of the HLA class I allele based on the signal pattern detected at the step (c) according to the Typing Table.
The PCR amplification of the target gene at the step (a), can be classified into 2 steps. One is a step to non-selectively amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles by the PCR method using a primer pair which can amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles. The other is a step to selectively amplify the specific group consisting of the specific HLA-A allele group or the specific HLA-B allele group by the PCR method using a primer pair which is specific to the common sequences to alleles of the specific group consisting of the specific HLA-A alleles or the specific HLA-B alleles. At the former step, PCR primers are designed to be specific to the common sequences, which are within the region of all alleles belonging to the HLA-A allele, the HLA-B allele or the HLA-C allele, or ahead and behind the region. At the latter step, PCR primers are designed to be specific to the common sequences to all alleles included in the specific group in order to amplify the specific group. When the specific group is selectively amplified in the presence of some groups, the primers described above don""t need to be used for both a sense primer and an anti sense primer of a primer pair corresponding to the specific group. It is possible to use the specific primer to the specific group for one of primers and the specific primer to all the groups for the other. The latter step can be performed according to the reference described by the inventors (Tissue Antigens 1997, Vol.50, 535-545). A method to selectively amplify alleles encoding the HLA-A2 antigen or the HLA-B40 antigen as a group is disclosed in the present description.
At the step (a), the PCR-amplified products derived from the allele belonging to the HLA-A alleles, the HLA-B alleles or the HLA-C alleles, or from the specific group, are produced. But it is not possible to distinguish the type of the HLA class I allele at the step. The hybridization reaction at the step (b) using the specific DNA probes is applied to the following steps.
The Typing Table at the step (d) is made using signal patterns obtained by hybridizing the PCR amplified products from samples whose HLA class I antigen types or allele types are known, with DNA probes which can specifically hybridize with the sequence of at least one specific HLA class I allele. Persons skilled in the art can make easily the Typing Table. As the Typing Table, FIGS. 1 to 6 can be referred. If someone wants to use DNA probes, which are not described in this description, another Typing Table can be used. The Typing Table is made from signal patterns obtained by hybridizing the PCR amplified products from samples whose HLA class I antigen types or allele types are known, with another DNA probe. As described above, persons skilled in the art can also make easily these Typing Tables. It should be considered that each sample has the HLA class I allele type in a homozygous or heterozygous state, when the HLA class I allele type is distinguished according to the Typing Tables.
In a perferable embodiment, the PCR method at the step (a) is performed by using a primer pair in which at least one of them is labeled, in order to detect whether or not the amplified products hybridize with immobilized DNA probes as signals at the step (c) described above. In the other embodiment, the above PCR can be performed by using 4 kinds of deoxyribonucleotide triphosphate (dNTP) in which at least one of them is labeled. As a substance used for labeling, a radioisotopic substance, or a non-radioisotopic substance such as a biotin or a digoxigenin, can be utilized.
In a preferable embodiment, at the step (b) or (c) described above, the hybridization of the products amplified by the PCR method with the immobilized DNA probe is performed by addiing an enzyme-conjugate which can specifically bond to a label of the amplified products is added at the same time as hybridization or after, and the amplified products hybridizing with the immobilized DNA probe is detected as signals by adding a chromogenic substrate, a luminescent substrate or a fluorescent substrate which can specifically react with the enzyme. When a peroxidase-conjugated streptavidin is used as an enzyme-conjugate, the signal can be immediately detected after washing by adding an enzyme-conjugate at the same time as hybridization.
In a preferable embodiment, at least one of a primer pair at the step (a) described above is biotinylated, and an enzyme-conjugate which can specifically bond to the biotinylated label at the step (b) or (c) is an enzyme-conjugated streptavidin, for example, a peroxydase-conjugated streptavidin or an alkaline phosphatase-conjugated streptavidin.
In a preferable embodiment, the hybridization of the products amplified by the PCR method with immobilized DNA probes is performed in a solution containing formamide at the step (b) described above. The formamide concentration of the solution described above (hybridization buffer) is from 5% to 30%, and from 10% to 25% as a preferable concentration. The concentration can be changed according to the sequence, the length and the type of the used DNA probe. The most preferable formamide concentration is about 20%.
In a preferable embodiment, the hybridization at the step (b) is performed in a solution containing formamide at the temperature of the 37xc2x0 C. The preferable temperature is from 32xc2x0 C. to 42xc2x0 C. The temperature can be changed according to the sequence, the length and the type of the used DNA probe as mentioned above for the formamide concentration. The most desirable temperature is about 37xc2x0 C. Hybridization is usually performed at comparatively high temperature, at about 65xc2x0 C., to improve the specificity. By using the solution containing formamide, the reaction can be performed at low temperature, at about 37xc2x0 C.
In a preferable embodiment, when the solution containing formamide is used for the hybridization at the step (b) described above, the temperature for washing after hybridization of the amplified products by the PCR method with immobilized DNA probes and/or after binding a label of the amplified products with an enzyme-conjugate is performed at room temperature. Namely, washing can be performed at low temperaure like room temperature as by using the solution containing formamide, as well as the above hybridization.
The amino-modified DNA which can specifically hybridize with at least one specific HLA-A allele, used at the step (b) in this invention, can be selected from the group consisting of A98T (SEQ ID No.:1), A98A (SEQ ID No.:2), A160A (SEQ ID No.:3), A239A (SEQ ID No.:4), A238A (SEQ ID No.:5), A240T (SEQ ID No.:6), A257TC (SEQ ID No.:7), A259AC (SEQ ID No.:8), A270T (SEQ ID No.:9), A282C (SEQ ID No.:10), A290T (SEQ ID No.:11), A299T (SEQ ID No.:12), A302G (SEQ ID No.:13), A355G (SEQ ID No.:14), A362TA (SEQ ID No.:15), A362TT (SEQ ID No.:16), A368A (SEQ ID No.:17), A368G (SEQ ID No.:18), A368T (SEQ ID No.:19), A402G (SEQ ID No.:20), A423T (SEQ ID No.:21), A448C (SEQ ID No.:22), A485A (SEQ ID No.:23), A524G (SEQ ID No.:24), A526T (SEQ ID No.:25), A527A (SEQ ID No.:26), A538CG (SEQ ID No.:27), A539A (SEQ ID No.:28), A539T (SEQ ID No.:29), A555T (SEQ ID No.:30), A559G (SEQ ID No.:31), A570CG (SEQ ID No.:32), A570GT (SEQ ID No.:33), A779A (SEQ ID No.:34), A843A (SEQ ID No.:35), A34 (SEQ ID No.:100), A282CT (SEQ ID No.:101), A290TR (SEQ ID No.:102), A302GR (SEQ ID No.:103), A414A (SEQ ID No.:104), A468T (SEQ ID No.:105), A489A (SEQ ID No.:106), A502C (SEQ ID No.:107), A538TG (SEQ ID No.:108) and complementary strands thereof.
The amino-modified DNA probe which can specifically hybridize with at 10 least one specific HLA-B allele can be selected from the group consisting of BL1 (SEQ ID No.:36), BL3 (SEQ ID No.:37), BL4 (SEQ ID No.:38), BL5 (SEQ ID No.:39), BL9 (SEQ ID No.:40), BL10 (SEQ ID No.:41), BL11 (SEQ ID No.:42), BL24 (SEQ ID No.:43), BL25 (SEQ ID No.:44), BL34 (SEQ ID No.:45), BL35 (SEQ ID No.:46), BL36 (SEQ ID No.:47), BL37 (SEQ ID No.:48), BL38 (SEQ ID No.:49), BL39 (SEQ ID No.:50), BL40 (SEQ ID No.:51), BL41 (SEQ ID No.:52), BL42 (SEQ ID No.:53), BL56 (SEQ ID No.:54), BL57 (SEQ ID No.:55), BL78 (SEQ ID No.:56), BL79 (SEQ ID No.:57), BL222A (SEQ ID No.:58), BL272GA (SEQ ID No.:59), BL226G (SEQ ID No.:60), BL292G (SEQ ID No.:61), BL292T (SEQ ID No.:62), BL361G (SEQ ID No.:63), BL409T (SEQ ID No.:64), BL512T (SEQ ID No.:65), BL538CG (SEQ ID No.:66), BL538G (SEQ ID No.:67), BL39R (SEQ ID No.:109), BL50 (SEQ ID No.:110), BL77 (SEQ ID No.:111), BL272A (SEQ ID No.:112), BL263T (SEQ ID No.:113), BL527A (SEQ ID No.:114), BL570GT (SEQ ID No.:115) and complementary strands thereof.
The amino-modified DNA probe which can specifically hybridize with at least one specific HLA-C allele can be selected from the group consisting of CC (SEQ ID No.:68), A-12 (SEQ ID No.:69), A-2 (SEQ ID No.:70), A-3 (SEQ ID No.:71), A-4 (SEQ ID No.:72), A-54 (SEQ ID No.:73), B-1 (SEQ ID No.:74), B-2 (SEQ ID No.:75), C-12 (SEQ ID No.:76), C-24 (SEQ ID No.:77), C-33 (SEQ ID No.:78), C-43 (SEQ ID No.:79), 134-g (SEQ ID No.:80), 134-A2 (SEQ ID No.:81), 353TCA1 (SEQ ID No.:82), 343A (SEQ ID No.:83), RA-2 (SEQ ID No.:116), RA-41 (SEQ ID No.:117), RB-28 (SEQ ID No.:118), 201g1 (SEQ ID No.:119), C206gR (SEQ ID No.:120), R341A (SEQ ID No.:121), R343g3 (SEQ ID No.:122), 353TCC (SEQ ID No.:123), 361T1 (SEQ ID No.:124), 361T368g (SEQ ID No.:125), 361T368T1 (SEQ ID No.:126), 369C (SEQ ID No.:127), 387g1 (SEQ ID No.:128), 526AC2 (SEQ ID No.:129), 538gAC (SEQ ID No.: 130) and complementary strands thereof.
This invention also comprises the DNA probe itself (from SEQ ID No.: 1 to SEQ ID No.:83 and from SEQ ID No.:100 to SEQ ID No.:130) which can specifically hybridize with at least one specific HLA-A allele, at least one specific HLA-B allele or at least one specific HLA-C allele for using the method for distinguishing the HLA class I allele type.
Both an amino-modified DNA probe and an unmodified DNA probe can be used. However, when the probe is covalently immobilized on wells of carboxylate-modified microtiter plates, the amino-modified probe must be used. Some bases can be deleted from or added to the end of the DNA probe within the range that the DNA probe can specifically hybridize with at least one specific HLA-A allele, at least one specific HLA-B allele or at least one specific HLA-C allele, namely, within the range that the DNA probe can keep the original specificity of hybridization. Accordingly, the DNA probes in this invention also comprise DNA probes wtherein some bases are deleted from or added to the nucleic acid sequence from SEQ ID No.:1 to SEQ ID No.:83 and SEQ ID No.:100 to SEQ ID No.:130 within the range described above.
The primers which can amplify all the HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles at the step (a) in this invention, can be selected from the group consisting of CGA011 (SEQ ID No.:90), CGA012 (SEQ ID No.:91), AIn3-66C (SEQ ID No.:92), 5BCIn37-34C (SEQ ID No.:96), 5BCIn37-24g (SEQ ID No.:97) and 5BCIn37-34g2 (SEQ ID No.:99). The primer which is specific to the common sequence to alleles of the specific group consisting of the specific HLA-A alleles or the specific HLA-B alleles, can be selected from A2-5T (SEQ ID No.:84), A3-273T (SEQ ID No.:85), A4-8C (SEQ ID No.:86), A4-254G (SEQ ID No.:87), BASF-1 (SEQ ID No.:88), and BASR-1 (SEQ ID No.:89). This invention comprises the primer itself described above (from SEQ ID No.:88 to SEQ ID No.:92, from SEQ ID No.:96 to SEQ ID No.:97 and SEQ ID No.:99), used for the method to type the HLA class I alleles.
Novel HLA-A alleles, HLA-B alleles and HLA-C alleles have been discovered. In the report of the WHO (World Health Organization) Nomenclature Committee for the HLA system, 82, 186, and 42 of alleles have been assigned for the HLA-A, -B and -C loci, respectively, at March 1997. This invention can discriminate all these alleles. Furthermore, the method shown in this invention, together with an optional, easy-performed improvement, such as adding extra DNA probes or primers, can cope with discrimination of alleles which may be discovered and enrolled in the future.
This invention can provide a kit and a reagent for typing of the HLA class I alleles described in this description. Furthermore, this invention can provide a kit and a reagent which comprise the DNA probes and the primers described in this description. For example, the kit can comprises a solution containing the primers (from SEQ ID No.:84 to SEQ ID No.:92, from SEQ ID No.:96 to SEQ ID No.:97 and SEQ ID No.:99) which is disclosed in this invention, PCR buffer solution, which may be concentrated solution, dNTPs, thermostable DNA polymerase, the DNA probes (from SEQ ID No.:84 to SEQ ID No.:92, from SEQ ID No.:96 to SEQ ID No.:97 and SEQ ID No.:99) which is disclosed in this invention or a microtiter plate on whose wells the DNA probes are covalently immobilized, a denature solution, a hybridization buffer, a washing solution and an instruction for the kit which includes the Typing Tables. The primer described above can optionally be labeled with a radioisotopic or non-radioisotopic substance. The primers can form a primer pair. The solution containing the primer can be freeze-dried. When the primer is not labeled, at least one of four kinds of dNTPs must be labeled. When a non-radioisotopic substance is used as a label, an enzyme-conjugate solution, a chromogenic reagent including a chromogenic substrate and a chromogenic solution, a luminescent reagent or a fluorescent reagent, a stop solution and so on can be added as a component in the kit. Furthermore, a component such as guanidine thiocyanate buffer for isolation of genome DNAs, can be optionally added in the kit to the degree promoting enforcement of this invention.